Abstract Hematopoietic stem and progenitor cell (HSPC) homeostasis is regulated by intricate cytokine receptor and tyrosine kinase signaling pathways. Janus Kinase 2 (JAK2) is the key tyrosine kinase in the signaling pathway of an array of hematopoietic receptors, including thrombopoietin (TPO) receptor (MPL) in hematopoietic stem cells (HSCs) and granulocyte macrophage colony-stimulating factor receptor (GM-CSFR) in myeloid progenitors. While JAK2 plays an essential role in hematopoietic development, uncontrolled JAK2 signaling results in hematopoietic malignancies. Gain-of-function JAK2 mutations such as V617F are found in large populations of patients with myeloproliferative neoplasms (MPNs), a clonal HSC disease. Genetically targeting JAK2 abrogates MPN in mice; the JAK2V617F mutation is the driving mutation found in the HSCs of human MPN patients. However, current FDA-approved JAK kinase inhibitors have low curative potential, indicating the need for a better understanding of the regulation of JAK2 for efficient targeting. A number of E3 ubiquitin ligases for JAK2 have been proposed, but none of them, when lost in vivo, increases JAK2 protein level in HSPCs, expands HSC pool, or enhances multiple lineage hematopoiesis. Thus, the E3 ligase(s) regulating JAK2 turnover remain elusive. Through a series of biochemical and functional studies, we found that JAK2 stability is regulated by the CBL family E3 ubiquitin ligases, c-CBL and CBL-b, via the adaptor protein LNK (also called SH2B3). C-Cbl-/- mice phenocopy Lnk-/- mice, exhibiting an augmented HSPC pool with superior transplantability and hypersensitivity to cytokines. Importantly, CBL loss-of-function mutations have been found in a wide range of myeloid malignancies with the most frequent occurrence in chronic and juvenile myelomonocytic leukemia (CMML and JMML), both of which bear poor prognosis. Here, we propose to define the molecular basis underlying the regulation of JAK2 stability and signaling through this novel signaling axis, CBL/LNK/JAK2, and exploit it for therapeutic strategies in treating myeloid neoplasms. The following specific aims will be addressed: 1. Investigate mechanisms by which CBL regulates JAK2 ubiquitination, stability and signaling in hematopoietic cell lines and HSPCs from Cbl deficient and Cbl E3 ligase inactive mouse models. 2. Determine the influence of CBL on the stability of constitutively active JAK2 mutants and mutant JAK2- mediated MPN development. 3. Determine the role of CBL in regulating JAK2 level and signaling in primary human progenitors and explore therapeutic potential of JAK inhibition in treating murine and human myeloid malignancies with CBL mutations. Collectively, our data point to a novel direct role of CBL-mediated JAK2 ubiquitination and degradation in cytokine signaling and hematopoiesis. The proposed studies will likely provide mechanistic insights into the regulation of wild type JAK2 in normal HSPCs as well as JAK2 mutants in MPNs. In addition, our studies may reveal novel therapeutic strategies for the treatment of CBLmut myeloid malignancies with poor prognosis.